Surface CD52, CD84, and PTGER2 mark mature PMN-MDSCs from cancer patients and G-CSF-treated donors.

Precise molecular characterization of circulating polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) is hampered by their mixed composition of mature and immature cells and lack of specific markers. Here, we focus on mature CD66b+CD10+CD16+CD11b+ PMN-MDSCs (mPMN-MDSCs) from either cancer patients or healthy donors receiving G-CSF for stem cell mobilization (GDs). By RNA sequencing (RNA-seq) experiments, we report the identification of a distinct gene signature shared by the different mPMN-MDSC populations under investigation, also validated in mPMN-MDSCs from GDs and tumor-associated neutrophils (TANs) by single-cell RNA-seq (scRNA-seq) experiments. Analysis of such a gene signature uncovers a specific transcriptional program associated with mPMN-MDSC differentiation and allows us to identify that, in patients with either solid or hematologic tumors and in GDs, CD52, CD84, and prostaglandin E receptor 2 (PTGER2) represent potential mPMN-MDSC-associated markers. Altogether, our findings indicate that mature PMN-MDSCs distinctively undergo specific reprogramming during differentiation and lay the groundwork for selective immunomonitoring, and eventually targeting, of mature PMN-MDSCs.

Human CD34+/dim neutrophil-committed progenitors do not differentiate into neutrophil-like CXCR1+CD14+CD16- monocytes in vitro.

The advent of recent cutting-edge technologies has allowed the discovery and characterization of novel progenitors of human neutrophils, including SSCloCD66b+CD15+CD11b-CD49dhiproNeu1s, SSChiCD66b+CD15+CD11b-CD49dintproNeus2s, CD66b+CD15+CD11b+CD49d+CD101-preNeus, and Lin-CD66b+CD117+CD71+eNePs. In this research field, we recently identified CD66b-CD38+CD64dimCD115-, CD34+, and CD34dim/- cells exclusively committed to the neutrophil lineage (which we renamed as CD34+ and CD34dim/- neutrophil-committed progenitors), representing the earliest neutrophil precursors identifiable and sorted by flow cytometry. Moreover, based on their differential CD34 and CD45RA expression, we could identify 4 populations of neutrophil-committed progenitors: CD34+CD45RA-/NCP1s, CD34+CD45RA+/NCP2s, CD34dim/-CD45RA+/NCP3s, and CD34dim/-CD45RA-/NCP4s. This said, a very recent study by Ikeda and coworkers (PMID: 36862552) reported that neutrophil precursors, termed either neutrophil progenitors or "early neutrophil-committed progenitors," would generate immunosuppressive neutrophil-like CXCR1+CD14+CD16- monocytes. Hence, presuming that neutrophil progenitors/"early neutrophil-committed progenitors" correspond to neutrophil-committed progenitors, the selective neutrophil commitment that we attributed to neutrophil-committed progenitors is contradicted by Ikeda and coworkers' article. In this study, by performing a more analytical reevaluation at the phenotypic and molecular levels of the cells generated by neutrophil-committed progenitors 2 and 4 (selected as representatives of neutrophil-committed progenitors), we categorically exclude that neutrophil-committed progenitors generate neutrophil-like CXCR1+CD14+CD16- monocytes. Rather, we provide substantial evidence indicating that the cells generated by neutrophil progenitors/"early neutrophil-committed progenitors" are neutrophilic cells at a different stage of maturation, displaying moderate levels of CD14, instead of neutrophil-like CXCR1+CD14+CD16- monocytes, as pointed by Ikeda and coworkers. Hence, the conclusion that neutrophil progenitors/"early neutrophil-committed progenitors" aberrantly differentiate into neutrophil-like monocytes derives, in our opinion, from data misinterpretation.

Plasmacytoid Dendritic Cell, Slan+-Monocyte and Natural Killer Cell Counts Function as Blood Cell-Based Biomarkers for Predicting Responses to Immune Checkpoint Inhibitor Monotherapy in Non-Small Cell Lung Cancer Patients.

The advent of immune checkpoint inhibitors (ICIs), for instance, programmed cell death 1 (PD-1)/PD-1 ligand 1 (PD-L1) blockers, has greatly improved the outcome of patients affected by non-small cell lung cancer (NSCLC). However, most NSCLC patients either do not respond to ICI monotherapy or develop resistance to it after an initial response. Therefore, the identification of biomarkers for predicting the response of patients to ICI monotherapy represents an urgent issue. Great efforts are currently dedicated toward identifying blood-based biomarkers to predict responses to ICI monotherapy. In this study, more commonly utilized blood-based biomarkers, such as the neutrophil-to-lymphocyte ratio (NLR) and the lung immune prognostic index (LIPI) score, as well as the frequency/number and activation status of various types of circulating innate immune cell populations, were evaluated in NSCLC patients at baseline before therapy initiation. The data indicated that, among all the parameters tested, low plasmacytoid dendritic cell (pDC), slan+-monocyte and natural killer cell counts, as well as a high LIPI score and elevated PD-L1 expression levels on type 1 conventional DCs (cDC1s), were independently correlated with a negative response to ICI therapy in NSCLC patients. The results from this study suggest that the evaluation of innate immune cell numbers and phenotypes may provide novel and promising predictive biomarkers for ICI monotherapy in NSCLC patients.

slan+ Monocytes Kill Cancer Cells Coated in Therapeutic Antibody by Trogoptosis.

Monocytes positive for 6-Sulfo LacNAc (slan) are a major subset of nonclassical CD14dimCD16+ monocytes in humans. We have shown that slan+ cells infiltrate lymphomas and elicit an antibody-dependent cellular cytotoxicity (ADCC) of neoplastic B cells mediated by the anti-CD20 therapeutic rituximab. Herein, by performing blocking experiments and flow cytometry analyses, as well as confocal microscopy and live-cell imaging assays, we extended the findings to other humanized antibodies and deciphered the underlying effector mechanism(s). Specifically, we show that, after coculture with target cells coated with anti-CD20 or anti-CD38, slan+ monocytes mediate trogocytosis, a cell-cell contact dependent, antibody-mediated process that triggers an active, mechanic disruption of target cell membranes. Trogocytosis by slan+ monocytes leads to a necrotic type of target cell death known as trogoptosis, which, once initiated, was partially sustained by endogenous TNFα. We also found that slan+ monocytes, unlike natural killer (NK) cells, mediate a direct ADCC with all types of anti-CD47 analyzed, and this was independent of their IgG isotype. The latter findings unveil a potentially relevant contribution by slan+ monocytes in mediating the therapeutic efficacy of anti-CD47 in clinical practice, which could be particularly important when NK cells are exhausted or deficient in number. Overall, our observations shed new light on the cytotoxic mechanisms exerted by slan+ monocytes in antibody-dependent tumor cell targeting and advance our knowledge on how to expand our therapeutic arsenal for cancer therapy.

CD14+-Monocytes Exposed to Apolipoprotein CIII Express Tissue Factor.

Apolipoprotein CIII (ApoCIII) represents a key regulator of plasma lipid metabolism and a recognized risk factor for atherosclerosis and cardiovascular diseases. Beyond the regulation of lipoprotein trafficking, ApoCIII is also involved in endothelial dysfunction and monocyte recruitment related to atherothrombosis. With tissue factor (TF) being the primary initiator of the blood coagulation cascade, we hypothesized that ApoCIII-treated monocytes could express it. Hence, human CD14+-monocytes and autologous neutrophils were incubated with ApoCIII and sera from human subjects containing previously measured ApoCIII amounts. By RT-qPCR and ELISA, CD14+-monocytes, but not neutrophils, were found to show increased mRNA expression and production of TNFα, IL-1ß and IL-6 as well as TF mRNA once exposed to ultra-purified ApoCIII. By flow cytometry, CD14+-monocytes were found to rapidly express TF on their cell surface membrane when incubated with either ApoCIII or sera with known concentrations of ApoCIII. Finally, preincubation with specific ApoCIII-neutralizing antibodies significantly reduced the ability of most sera with known concentrations of ApoCIII to upregulate TF protein, other than partially inhibiting cytokine release, in CD14+-monocytes. In sum, herein we demonstrate that ApoCIII activates CD14+-monocytes to express TF. The data identify a potential mechanism which links circulating apolipoproteins with inflammation and atherothrombosis-related processes underlying cardiovascular risk.

Current knowledge on the early stages of human neutropoiesis.

Polymorphonuclear neutrophils are no longer considered as a homogeneous population of terminally differentiated and short-lived cells that belong to the innate immune system only. In fact, data from the past decades have uncovered that neutrophils exhibit large phenotypic heterogeneity and functional versatility that render them more plastic than previously thought. Hence, their precise role as effector cells in inflammation, in immune response and in other pathophysiological processes, including tumors, needs to be better evaluated. In such a complex scenario, common knowledge of the differentiation of neutrophils in bone marrow refers to lineage precursors, starting from the still poorly defined myeloblasts, and proceeding sequentially to promyelocytes, myelocytes, metamyelocytes, band cells, segmented neutrophils, and mature neutrophils, with each progenitor stage being more mature and better characterized. Thanks to the development and utilization of cutting-edge technologies, novel information about neutrophil precursors at stages earlier than the promyelocytes, hence closer to the hematopoietic stem cells, is emerging. Accordingly, this review discusses the main findings related to the very early precursors of human neutrophils and provides our perspectives on human neutropoiesis.

SARS-CoV-2-Associated ssRNAs Activate Human Neutrophils in a TLR8-Dependent Fashion.

COVID-19 disease is characterized by a dysregulation of the innate arm of the immune system. However, the mechanisms whereby innate immune cells, including neutrophils, become activated in patients are not completely understood. Recently, we showed that GU-rich RNA sequences from the SARS-CoV-2 genome (i.e., SCV2-RNA1 and SCV2-RNA2) activate dendritic cells. To clarify whether human neutrophils may also represent targets of SCV2-RNAs, neutrophils were treated with either SCV2-RNAs or, as a control, R848 (a TLR7/8 ligand), and were then analyzed for several functional assays and also subjected to RNA-seq experiments. Results highlight a remarkable response of neutrophils to SCV2-RNAs in terms of TNFα, IL-1ra, CXCL8 production, apoptosis delay, modulation of CD11b and CD62L expression, and release of neutrophil extracellular traps. By RNA-seq experiments, we observed that SCV2-RNA2 promotes a transcriptional reprogramming of neutrophils, characterized by the induction of thousands of proinflammatory genes, similar to that promoted by R848. Furthermore, by using CU-CPT9a, a TLR8-specific inhibitor, we found that SCV2-RNA2 stimulates neutrophils exclusively via TLR8-dependent pathways. In sum, our study proves that single-strand RNAs from the SARS-CoV-2 genome potently activate human neutrophils via TLR8, thus uncovering a potential mechanism whereby neutrophils may contribute to the pathogenesis of severe COVID-19 disease.

Neutrophils inhibit γδ T cell functions in the imiquimod-induced mouse model of psoriasis.

Background: Psoriasis is a chronic skin disease associated with deregulated interplays between immune cells and keratinocytes. Neutrophil accumulation in the skin is a histological feature that characterizes psoriasis. However, the role of neutrophils in psoriasis onset and development remains poorly understood. Methods: In this study, we utilized the model of psoriasiform dermatitis, caused by the repeated topical application of an imiquimod containing cream, in neutrophil-depleted mice or in mice carrying impairment in neutrophil functions, including p47phox -/- mice (lacking a cytosolic subunit of the phagocyte nicotinamide adenine dinucleotide phosphate - NADPH - oxidase) and Sykfl/fl MRP8-cre+ mice (carrying the specific deletion of the Syk kinase in neutrophils only), to elucidate the specific contribution of neutrophils to psoriasis development. Results: By analyzing disease development/progression in neutrophil-depleted mice, we now report that neutrophils act as negative modulators of disease propagation and exacerbation by inhibiting gammadelta T cell effector functions via nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-mediated reactive oxygen species (ROS) production. We also report that Syk functions as a crucial molecule in determining the outcome of neutrophil and γδ T cell interactions. Accordingly, we uncover that a selective impairment of Syk-dependent signaling in neutrophils is sufficient to reproduce the enhancement of skin inflammation and γδ T cell infiltration observed in neutrophil-depleted mice. Conclusions: Overall, our findings add new insights into the specific contribution of neutrophils to disease progression in the IMQ-induced mouse model of psoriasis, namely as negative regulatory cells.

The PDE4 Inhibitor Tanimilast Restrains the Tissue-Damaging Properties of Human Neutrophils.

Neutrophils, the most abundant subset of leukocytes in the blood, play a pivotal role in host response against invading pathogens. However, in respiratory diseases, excessive infiltration and activation of neutrophils can lead to tissue damage. Tanimilast-international non-proprietary name of CHF6001-is a novel inhaled phosphodiesterase 4 (PDE4) inhibitor in advanced clinical development for the treatment of chronic obstructive pulmonary disease (COPD), a chronic inflammatory lung disease where neutrophilic inflammation plays a key pathological role. Human neutrophils from healthy donors were exposed to pro-inflammatory stimuli in the presence or absence of tanimilast and budesonide-a typical inhaled corticosteroid drug-to investigate the modulation of effector functions including adherence to endothelial cells, granule protein exocytosis, release of extracellular DNA traps, cytokine secretion, and cell survival. Tanimilast significantly decreased neutrophil-endothelium adhesion, degranulation, extracellular DNA traps casting, and cytokine secretion. In contrast, it promoted neutrophil survival by decreasing both spontaneous apoptosis and cell death in the presence of pro-survival factors. The present work suggests that tanimilast can alleviate the severe tissue damage caused by massive recruitment and activation of neutrophils in inflammatory diseases such as COPD.

CD66b-CD64dimCD115- cells in the human bone marrow represent neutrophil-committed progenitors.

Here we report the identification of human CD66b-CD64dimCD115- neutrophil-committed progenitor cells (NCPs) within the SSCloCD45dimCD34+ and CD34dim/- subsets in the bone marrow. NCPs were either CD45RA+ or CD45RA-, and in vitro experiments showed that CD45RA acquisition was not mandatory for their maturation process. NCPs exclusively generated human CD66b+ neutrophils in both in vitro differentiation and in vivo adoptive transfer experiments. Single-cell RNA-sequencing analysis indicated NCPs fell into four clusters, characterized by different maturation stages and distributed along two differentiation routes. One of the clusters was characterized by an interferon-stimulated gene signature, consistent with the reported expansion of peripheral mature neutrophil subsets that express interferon-stimulated genes in diseased individuals. Finally, comparison of transcriptomic and phenotypic profiles indicated NCPs represented earlier neutrophil precursors than the previously described early neutrophil progenitors (eNePs), proNeus and COVID-19 proNeus. Altogether, our data shed light on the very early phases of neutrophil ontogeny.

SARS-CoV-2-associated ssRNAs activate inflammation and immunity via TLR7/8.

The inflammatory and IFN pathways of innate immunity play a key role in the resistance and pathogenesis of coronavirus disease 2019 (COVID-19). Innate sensors and SARS-CoV-2-associated molecular patterns (SAMPs) remain to be completely defined. Here, we identified single-stranded RNA (ssRNA) fragments from the SARS-CoV-2 genome as direct activators of endosomal TLR7/8 and MyD88 pathway. The same sequences induced human DC activation in terms of phenotype and function, such as IFN and cytokine production and Th1 polarization. A bioinformatic scan of the viral genome identified several hundreds of fragments potentially activating TLR7/8, suggesting that products of virus endosomal processing potently activate the IFN and inflammatory responses downstream of these receptors. In vivo, SAMPs induced MyD88-dependent lung inflammation characterized by accumulation of proinflammatory and cytotoxic mediators and immune cell infiltration, as well as splenic DC phenotypical maturation. These results identified TLR7/8 as a crucial cellular sensor of ssRNAs encoded by SARS-CoV-2 involved in host resistance and the disease pathogenesis of COVID-19.

Induction of OCT2 contributes to regulate the gene expression program in human neutrophils activated via TLR8.

The transcription factors (TFs) that regulate inducible genes in activated neutrophils are not yet completely characterized. Herein, we show that the genomic distribution of the histone modification H3K27Ac, as well as PU.1 and C/EBPß, two myeloid-lineage-determining TFs (LDTFs), significantly changes in human neutrophils treated with R848, a ligand of Toll-like receptor 8 (TLR8). Interestingly, differentially acetylated and LDTF-marked regions reveal an over-representation of OCT-binding motifs that are selectively bound by OCT2/POU2F2. Analysis of OCT2 genomic distribution in primary neutrophils and of OCT2-depletion in HL-60-differentiated neutrophils proves the requirement for OCT2 in contributing to promote, along with nuclear factor κB (NF-κB) and activator protein 1 (AP-1), the TLR8-induced gene expression program in neutrophils. Altogether, our data demonstrate that neutrophils, upon activation via TLR8, profoundly reprogram their chromatin status, ultimately displaying cell-specific, prolonged transcriptome changes. Data also show an unexpected role for OCT2 in amplifying the transcriptional response to TLR8-mediated activation.

Tumor-associated neutrophils (TANs) in human carcinoma-draining lymph nodes: a novel TAN compartment.

OBJECTIVES: The role of tumor-associated neutrophils (TANs) in the nodal spread of cancer cells remains unexplored. The present study evaluates the occurrence and clinical significance of human nodal TANs. METHODS: The relevance, derivation, phenotype and interactions of nodal TANs were explored via a large immunohistochemical analysis of carcinoma-draining lymph nodes, and their clinical significance was evaluated on a retrospective cohort of oral squamous cell carcinomas (OSCC). The tumor-promoting function of nodal TAN was probed in the OSCC TCGA dataset combining TAN and epithelial-to-mesenchymal transition (EMT) signatures. RESULTS: The pan-carcinoma screening identified a consistent infiltration (59%) of CD66b+  TANs in tumor-draining lymph nodes (TDLNs). Microscopic findings, including the occurrence of intra-lymphatic conjugates of TANs and cancer cells, indicate that TANs migrate through lymphatic vessels. In vitro experiments revealed that OSCC cell lines sustain neutrophil viability and activation via release of GM-CSF. Moreover, by retrospective analysis, a high CD66b+ TAN density in M-TDLNs of OSCC (n = 182 patients) predicted a worse prognosis. The analysis of the OSCC-TCGA dataset unveiled that the expression of a set of neutrophil-specific genes in the primary tumor (PT) is highly associated with an EMT signature, which predicts nodal spread. Accordingly, in the PT of OSCC cases, CD66b+TANs co-localised with PDPN+S100A9- EMT-switched tumor cells in areas of lymphangiogenesis. The pro-EMT signature is lacking in peripheral blood neutrophils from OSCC patients, suggesting tissue skewing of TANs. CONCLUSION: Our findings are consistent with a novel pro-tumoral TAN compartment that may promote nodal spread via EMT, through the lymphatics.

On the Improper Use of the Term High-Density Neutrophils.

Recent studies have revealed that neutrophils exhibit an unsuspected heterogeneity. In this context, the term high-density neutrophils (HDNs) has recently gained ground to define nothing more than neutrophils displaying an unaltered normal density. Therefore, as discussed here, we argue that the HDNs term must be avoided, as it is confounding and scientifically inappropriate.

The global response to the COVID-19 pandemic: how have immunology societies contributed?

The COVID-19 pandemic is shining a spotlight on the field of immunology like never before. To appreciate the diverse ways in which immunologists have contributed, Nature Reviews Immunology invited the president of the International Union of Immunological Societies and the presidents of 15 other national immunology societies to discuss how they and their members responded following the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Human neutrophils activated by TLR8 agonists, with or without IFNγ, synthesize and release EBI3, but not IL-12, IL-27, IL-35, or IL-39.

The IL-12 family of cytokines plays crucial functions in innate and adaptive immunity. These cytokines include heterodimers sharing distinct α (IL-12A, IL-23A, and IL-27A) with two ß (IL-12B and Epstein-Barr virus induced gene 3 [EBI3]) chains, respectively, IL-12 (IL-12B plus IL-12A) and IL-23 (IL-12B plus IL-23A) sharing IL-12B, IL-27 (EBI3 plus IL-27A), IL-35 (EBI3 plus IL-12A), and IL-39 (EBI3 plus IL-23A) sharing EBI3. In this context, we have recently reported that highly pure neutrophils incubated with TLR8 agonists produce functional IL-23. Previously, we showed that neutrophils incubated with LPS plus IFNγ for 20 h produce IL-12. Herein, we investigated whether highly pure, TLR8-activated, neutrophils produce EBI3, and in turn IL-27, IL-35, and IL-39, the IL-12 members containing it. We report that neutrophils incubated with TLR8 ligands, TNFα and, to a lesser extent, LPS, produce and release remarkable amounts of EBI3, but not IL-27A, consequently excluding the possibility for an IL-27 production. We also report a series of unsuccessful experiments performed to investigate whether neutrophil-derived EBI3 associates with IL-23A to form IL-39. Furthermore, we show that neutrophils incubated with IFNγ in combination with either TLR8 or TLR4 ligands express/produce neither IL-12, nor IL-35, due to the inability of IFNγ, contrary to previous findings, to activate IL12A transcription. Even IL-27 was undetectable in supernatants harvested from IFNγ plus R848-treated neutrophils, although they were found to accumulate IL27A transcripts. Finally, by immunohistochemistry experiments, EBI3-positive neutrophils were found in discrete pathologies only, including diverticulitis, cholecystitis, Gorham disease, and Bartonella Henselae infection, implying a specific role of neutrophil-derived EBI3 in vivo.

Fast and Accurate Quantitative Analysis of Cytokine Gene Expression in Human Neutrophils by Reverse Transcription Real-Time PCR.

Polymorphonuclear neutrophils, traditionally viewed as short-lived effector cells, are nowadays regarded as important components of effector and regulatory circuits in the innate and adaptive immune systems. Most of the physiological functions of neutrophils as crucial players in the host immune response, able not only to act in the early phases of acute inflammation but also to condition the progression of the inflammatory reaction and the subsequent initiation of the specific immune response, relies on their capacity to produce and release a number of proinflammatory and immunoregulatory cytokines. This fact has reevaluated the importance, the role, and the physiological and pathological significance of neutrophils in the pathogenesis of inflammatory, infectious, autoimmune, and neoplastic diseases and has identified neutrophils as an important potential target for selective pharmacological intervention to both promote and restrain inflammation. In this context, understanding the mechanisms of modulation of neutrophil-derived cytokines and chemokines represents a critical step toward a better understanding of how neutrophils may influence pathophysiological processes in vivo. Herein, we describe and discuss an updated version of the methods that we have developed to rapidly and precisely characterize the pattern of cytokine expression in in vitro-activated human neutrophils. The validation of the reverse transcription quantitative real-time PCR assay as a suitable strategy for an accurate, sensitive, reliable, and bona fide analysis of cytokine gene expression in human neutrophils overcomes several problems strictly specific to neutrophils and offers an important tool, in the neutrophil research area, to test many experimental conditions for gene expression analysis.

6-Sulfo LacNAc (Slan) as a Marker for Non-classical Monocytes.

Monocytes are subdivided into three subsets, which have different phenotypic and functional characteristics and different roles in inflammation and malignancy. When in man CD14 and CD16 monoclonal antibodies are used to define these subsets, then the distinction of non-classical CD14low and intermediate CD14high monocytes requires setting a gate in what is a gradually changing level of CD14 expression. In the search for an additional marker to better dissect the two subsets we have explored the marker 6-sulfo LacNAc (slan). Slan is a carbohydrate residue originally described to be expressed on the cell surface of a type of dendritic cell in human blood. We elaborate herein that the features of slan+ cells are congruent with the features of CD16+ non-classical monocytes and that slan is a candidate marker for definition of non-classical monocytes. The use of this marker may help in studying the role of non-classical monocytes in health and in diagnosis and monitoring of disease.